Women with the highest mammographic density are at a four- to six-fold increased risk of breast cancer as compared with women with the lowest mammographic density. The data examining the underlying mechanisms involved are sparse. The widely hypothesized belief that mammographic density reflects cumulative exposure to estrogens has come under question with data from our own group and others. Most studies to date have focused on postmenopausal women, whose levels of estrogens and breast density are both lower than those of premenopausal women. It is highly plausible that mammographic density may be more reflective of premenopausal hormone levels than postmenopausal levels. In addition, a number of other hormones including insulin-like growth factor-1 and vitamin D, and estrogen metabolites have also been implicated in breast proliferation and carcinogenesis and may be associated with breast density. In my first R01 submission, I propose to explore the underlying mechanism by which mammographic density is related to breast cancer, by focusing on circulating hormone levels and urinary estrogen metabolites in premenopausal women. Specifically, we will: (1) Assess the relationship between circulating levels of estrogens, androgens, prolactin, sex hormone binding globulin, vitamin D and insulin-like growth factor-1, and mammographic density;(2) Examine the association between urinary estrogen metabolites in relation to mammographic density;and (3) Determine if hormonal biomarkers and mammographic density are independent predictors of breast cancer risk. These aims will provide complementary information that will help elucidate the biology of mammographic density. We will collect film mammograms and measure breast density among women in a breast cancer case- control study nested within the Nurses'Health Study II for whom blood samples are available and hormone assays will be conducted as part of another funded project. This case-control study is unique in that sex steroid hormones have been measured in both the early follicular and mid-luteal phases of the menstrual cycle. Recent work from our group highlights important differences observed between phase-specific hormone levels and breast cancer risk. To date, no other study has examined phase-specific sex steroids in relation to mammographic density. We expect to have mammographic density as well as hormone measurements on approximately 636 breast cancer cases (361 with timed samples) and 2 matched controls per case. Measurements of both biomarkers and breast density may help to identify women at particularly high risk of breast cancer. This is an efficient study design to determine the association between hormonal biomarkers and mammographic density and the effects of both on breast cancer risk. PUBLIC HEALTH RELEVANCE: Mammographic density and circulating hormone levels are both well-established strong predictors of breast cancer risk. We propose to prospectively examine the association between premenopausal levels of circulating and urinary biomarkers and mammographic density, and determine if these biomarkers and breast density independently predict breast cancer risk. Understanding the relation between the two and how they influence breast cancer risk will be useful in risk prediction and may help to identify women at particularly high risk of breast cancer.